搜索结果: 'gmp t cell activator'

Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration,Stargardt disease,and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements,such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient,but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study,we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand,administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly,transfection of mRNA encoding a key regulator of RPE gene expression,microphthalmia-associated transcription factor (MITF),confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF,primarily localized in the nucleus. Despite these findings,quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings,therefore,show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe,efficient,and functional. View Publication

HIV-1 infection leads to numerous B cell abnormalities,including hypergammaglobulinemia,nonspecific B cell activation,nonspecific class switching,increased cell turnover,breakage of tolerance,increased immature/transitional B cells,B cell malignancies,as well as a loss of capacity to generate and maintain memory,all of which contribute to a global impairment of the immune humoral compartment. Several cytokines and soluble factors,which are increased in sera of HIV-1-infected individuals,have been suggested to directly or indirectly contribute to these B cell dysfunctions,and one of these is the B cell-activating factor (BAFF). We report in this study that HIV-1 (X4- and R5-tropic) upregulates BAFF expression and secretion by human monocytes. Moreover,we show that the virus-mediated production of BAFF by monocytes relies on a type I IFN response by a small percentage of plasmacytoid dendritic cells (pDCs) present in the monocyte cultures. HIV-1-induced type I IFN by pDCs triggers BAFF production in both classical and intermediate monocytes,but not in nonclassical monocytes,which nonetheless display a very strong basal BAFF production. We report also that basal BAFF secretion was higher in monocytes obtained from females compared with those from male donors. This study provides a novel mechanistic explanation for the increased BAFF levels observed during HIV-1 infection and highlights the importance of pDC/monocyte crosstalk to drive BAFF secretion. View Publication

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes,smooth muscle cells (SMC),and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs,differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result,numerous strategies have been developed to derive CPCs from ESCs. In this protocol,differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs,ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus,CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs. View Publication

UNLABELLED In the classical form of $\$1-antitrypsin deficiency (ATD),aberrant intracellular accumulation of misfolded mutant $\$1-antitrypsin Z (ATZ) in hepatocytes causes hepatic damage by a gain-of-function,proteotoxic" mechanism. Whereas some ATD patients develop severe liver disease (SLD) that necessitates liver transplantation� View Publication

Amyotrophic lateral sclerosis is a progressive disease characterized by the loss of upper and lower motor neurons,leading to paralysis of voluntary muscles. About 10% of all ALS cases are familial (fALS),among which 15-20% are linked to Cu/Zn superoxide dismutase (SOD1) mutations,usually inherited in an autosomal dominant manner. To date only one FDA approved drug is available which increases survival moderately. Our understanding of ALS disease mechanisms is largely derived from rodent model studies,however due to the differences between rodents and humans,it is necessary to have humanized models for studies of disease pathogenesis as well as drug development. Therefore,we generated a comprehensive library of a total 22 of fALS patient-specific induced pluripotent stem cell (iPSC) lines. These cells were thoroughly characterized before being deposited into the library. The library of cells includes a variety of C9orf72 mutations,sod1 mutations,FUS,ANG and FIG4 mutations. Certain mutations are represented with more than one line,which allows for studies of variable genetic backgrounds. In addition,these iPSCs can be successfully differentiated to astroglia,a cell type known to play a critical role in ALS disease progression. This library represents a comprehensive resource that can be used for ALS disease modeling and the development of novel therapeutics. View Publication

It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study,we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors,by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner,which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors,in a 2-dimensional or 3-dimensional environment. However,Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices,we observed mature Purkinje-like cells with right morphology and marker expression patterns,which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases. View Publication

Inactivation of the Pancreatic and Duodenal Homeobox 1 (PDX1) gene causes pancreatic agenesis,which places PDX1 high atop the regulatory network controlling development of this indispensable organ. However,little is known about the identity of PDX1 transcriptional targets. We simulated pancreatic development by differentiating human embryonic stem cells (hESCs) into early pancreatic progenitors and subjected this cell population to PDX1 chromatin immunoprecipitation sequencing (ChIP-seq). We identified more than 350 genes bound by PDX1,whose expression was upregulated on day 17 of differentiation. This group included known PDX1 targets and many genes not previously linked to pancreatic development. ChIP-seq also revealed PDX1 occupancy at hepatic genes. We hypothesized that simultaneous PDX1-driven activation of pancreatic and repression of hepatic programs underlie early divergence between pancreas and liver. In HepG2 cells and differentiating hESCs,we found that PDX1 binds and suppresses expression of endogenous liver genes. These findings rebrand PDX1 as a context-dependent transcriptional repressor and activator within the same cell type. View Publication

While enucleation is a critical step in the terminal differentiationbackslashnof human red blood cells,the molecular mechanisms underlying thisbackslashnunique process remain unclear. To investigate erythroblast enucleationbackslashnwe studied the erythroid differentiation of human embryonic stembackslashncells (hESCs),which provide a unique model for deeper understandingbackslashnof the development and differentiation of multiple cell types. Firstly,backslashnusing a two-step protocol,we demonstrated that terminal erythroidbackslashndifferentiation from hESCs is directly dependent on the age of thebackslashnembryoid bodies. Secondly,by choosing hESCs in two extreme conditionsbackslashnof erythroid culture,we obtained an original differentiation modelbackslashnwhich allows one to study the mechanisms underlying the enucleationbackslashnof erythroid cells by analyzing the gene and miRNA (miR) expressionbackslashnprofiles of cells from these two culture conditions. Thirdly,usingbackslashnan integrated analysis of mRNA and miR expression profiles,we identifiedbackslashn5 miRs potentially involved in erythroblast enucleation. Finally,backslashnby selective knockdown of these 5 miRs we found miR-30a to be a regulatorbackslashnof erythroblast enucleation in hESCs. This article is protected bybackslashncopyright. All rights reserved. View Publication

Epigenetic modifications refer to a number of biological processes which alter the structure of chromatin and its transcriptional activity such as DNA methylation and histone post-translational processing. Studies have tried to elucidate how the viral genome and its products are affected by epigenetic modifications imposed by cell machinery and how it affects the ability of the virus to either,replicate and produce a viable progeny or be driven to latency. The purpose of this study was to evaluate epigenetic modifications in PBMCs and CD4+ cells after HIV-1 infection analyzing three approaches: (i) global DNA- methylation; (ii) qPCR array and (iii) western blot. HIV-1 infection led to methylation increases in the cellular DNA regardless the activation status of PBMCs. The analysis of H3K9me3 and H3K27me3 suggested a trend towards transcriptional repression in activated cells after HIV-1 infection. Using a qPCR array,we detected genes related to epigenetic processes highly modulated in activated HIV-1 infected cells. SETDB2 and RSK2 transcripts showed highest up-regulation levels. SETDB2 signaling is related to transcriptional silencing while RSK2 is related to either silencing or activation of gene expression depending on the signaling pathway triggered down-stream. In addition,activated cells infected by HIV-1 showed lower CD69 expression and a decrease of IL-2,IFN-γ and metabolism-related factors transcripts indicating a possible functional consequence towards global transcriptional repression found in HIV-1 infected cells. Conversely,based on epigenetic markers studied here,non-stimulated cells infected by HIV-1,showed signs of global transcriptional activation. Our results suggest that HIV-1 infection exerts epigenetic modulations in activated cells that may lead these cells to transcriptional repression with important functional consequences. Moreover,non-stimulated cells seem to increase gene transcription after HIV-1 infection. Based on these observations,it is possible to speculate that the outcome of viral infections may be influenced by the cellular activation status at the moment of infection. View Publication

BACKGROUND In colorectal carcinoma (CRC),activation of the Raf/MEK/ERK signaling pathway is commonly observed. In addition,the commonly used 5FU-based chemotherapy in patients with metastatic CRC was found to enrich a subpopulation of CD26(+) cancer stem cells (CSCs). As activation of the Raf/MEK/ERK signaling pathway was also found in the CD26(+) CSCs and therefore,we hypothesized that an ATP-competitive pan-Raf inhibitor,Raf265,is effective in eliminating the cancer cells and the CD26(+) CSCs in CRC patients. METHODS HT29 and HCT116 cells were treated with various concentrations of Raf265 to study the anti-proliferative and apoptotic effects of Raf265. Anti-tumor effect was also demonstrated using a xenograft model. Cells were also treated with Raf265 in combination with 5FU to demonstrate the anti-migratory and invasive effects by targeting on the CD26(+) CSCs and the anti-metastatic effect of the combined treatment was shown in an orthotopic CRC model. RESULTS Raf265 was found to be highly effective in inhibiting cell proliferation and tumor growth through the inhibition of the RAF/MEK/ERK signaling pathway. In addition,anti-migratory and invasive effect was found with Raf265 treatment in combination with 5FU by targeting on the CD26(+) cells. Finally,the anti-tumor and anti-metastatic effect of Raf265 in combination with 5FU was also demonstrated. CONCLUSIONS This preclinical study demonstrates the anti-tumor and anti-metastatic activity of Raf265 in CRC,providing the basis for exploiting its potential use and combination therapy with 5FU in the clinical treatment of CRC. View Publication

BACKGROUND Acute myeloid leukemia (AML) is initiated and maintained by a subset of self-renewing leukemia stem cells (LSCs),which contribute to the progression,recurrence and therapeutic resistance of leukemia. However,the mechanisms underlying the maintenance of LSCs drug resistance have not been fully defined. In this study,we attempted to elucidate the mechanisms of LSCs drug resistance. METHODS We performed reverse phase protein arrays to analyze the expression of anti-apoptotic proteins in the LSC-enriched leukemia cell line KG-1a. Immuno-blotting,cell viability and clinical AML samples were evaluated to verify the micro-assay results. The characteristics and transcriptional regulation of survivin were analyzed with the relative luciferase reporter assay,mutant constructs,chromatin immuno-precipitation (ChIP),quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR),and western blotting. The levels of Sp1,c-Myc,phospho-extracellular signal-regulated kinase (p-ERK),phospho-mitogen and stress-activated protein kinase (p-MSK) were investigated in paired CD34+ and CD34- AML patient samples. RESULTS Survivin was highly over-expressed in CD34 + CD38- KG-1a cells and paired CD34+ AML patients compared with their differentiated counterparts. Functionally,survivin contributes to the drug resistance of LSCs,and Sp1 and c-Myc concurrently regulate levels of survivin transcription. Clinically,Sp1 and c-Myc were significantly up-regulated and positively correlated with survivin in CD34+ AML patients. Moreover,Sp1 and c-Myc were further activated by the ERK/MSK mitogen-activated protein kinase (MAPK) signaling pathway,modulating survivin levels. CONCLUSION Our findings demonstrated that ERK/MSK/Sp1/c-Myc axis functioned as a critical regulator of survivin expression in LSCs,offering a potential new therapeutic strategy for LSCs therapy. View Publication

Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes,cells from the corneal stroma,may have the potential for restoration of vision in cell therapy and biomedical engineering applications,but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells,maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype. View Publication